The central objective of this project is to elucidate the fundamental factors that influence the function of the active sites of redox metalloproteins. These proteins are essential to all organisms. They are ubiquitous in processes where biological electron transfer occurs, for example in respiration, photosynthesis, activation of small molecules, and the oxygenation of organic substrates. Adequate understanding of the function of these proteins cannot be expected unless the principles governing the behavior of the active metal sites are understood. We propose to establish these principles for key systems having general significance. The principles may then be applied to redox metalloproteins as a general class. Key issues include the following. How do redox metalloproteins recognize, admit, and control the binding of substrate? How is substrate activation accomplished? How do the proteins carry out facile electron transfer, and how do they modify or switch electron transfer dynamics and thermodynamics in response to functional requirements and ambient conditions? How do they perform coupled functions such as energy storage by proton pumping? Our general approaches to these problems are various techniques in optical spectroscopy. We have previously emphasized vibrational spectroscopies, but our experience has shown that complex metalloprotein issues must be addressed by many different techniques. Accordingly this proposal is more general than its predecessors, and the title reflects this broadened emphasis. During the past grant period we have developed specific hypotheses as to how labile coordination chemistry ("ligand shuttling") at the metal centers of redox metalloproteins may play key roles in active site function. A significant fraction of this proposal is focused on testing these hypotheses using the heme-copper oxidases as target systems. We also propose studies that are directed at significant issues in other redox metalloproteins. These include application of novel far-red Raman technique to copper proteins having chromophores that absorb at very low energy (e.g. the nitrous oxide reductases) and infrared studies of the functional and photochemical reactions of nickel hydrogenases.